Method for labeling and detecting materials employing arylsulfonate cyanine dyes

ABSTRACT

A luminescent cyanine dye having generally the following structure ##STR1## wherein the dotted lines represent one to three rings having 5 to 6 atoms in each ring. R 3 , R 4 , R 8  and R 9  groups are attached to the rings. At least one of the R 8  and R 9  groups is a sulfonic acid or sulfonate group and at least one of the R 1 , R 2 , R 3 , R 4  and R 7  groups is a reactive moiety that reacts with amino, hydroxy or sulfhydryl nucleophiles.

This invention was made in part under National Institutes of Healthcontract numbers NIH-NS-19353 and NIH-GM-34639.

This application is a continuation of Ser. No. 240,756, filed Sep. 2,1988, which is a continuation-in-part of Ser. No. 854,347, filed Apr.18, 1986 both now abandoned.

BACKGROUND OF THE INVENTION

Cyanine and related polymethine dyes having light absorbing propertieshave been employed in photographic films. Although such dyes requirelight absorbing properties, they do not require luminescence(fluorescence or phosphorescence) properties. Cyanine dyes havingluminescence properties heretofore have had very limited utilization.One such utilization involved specifically labeling the sulfhydryl groupof proteins. In one report, Salama, G., Waggoner, A. S., and Abramson,J., have reported under the title Sulfhydryl Reagent Dyes Trigger theRapid Release of Ca²⁺ from Sarcoplasmic Reticulum Vesicles (SR),Biophysical Journal, 47, 456a (1985) that cyanine chromophores having aniodoacetyl group was used to form covalent bonds with sulfhydryl groupson the Sarcoplasmic Reticulum protein at pH 6.7 to trigger Ca²⁺ release.The report also stated that fluorescent dyes were used to label andisolate those proteins.

In a report of Waggoner, A. S., Jenkins, P. L., Carpenter, J. P., andGupta, R., entitled The Kinetics of Conformational Changes in a Regionof the Rhodopsin Molecule Away From the Retinylidene Binding Site,Biophysical Journal, 33, 292a (1981), the authors state that thesulfhydryl group on the F1 region of cattle rhodopsin has beencovalently labeled with a cyanine dye having absorbance at 660 nm.Again, this report used cyanine dyes for labeling specifically thesulfhydryl group of a protein, but does not disclose that fluorescentdyes were used.

An article entitled International Workshop on the Application ofFluorescence Photobleaching Techniques to Problems in Cell Biology,Jacobson K., Elson E., Koppel D., Webb W., Fed. Proc. 42:72-79 (1983),reports on a paper delivered by A. Waggoner relating to cyanine-typefluorescent probes which can be conjugated to proteins and can beexcited in the deeper red region of the spectrum.

The only cyanine probes mentioned in any of the above three reports arethose which covalently attach specifically to the sulfhydryl group of aprotein. The only specific cyanine compound mentioned is one having aniodoacetyl group, which group causes the cyanine dye to be covalentlyreactive with a sulfhydryl group. None of the articles listed abovediscloses the covalent reaction of a cyanine dye with any material otherthan a protein or with any group on a protein other than a sulfhydrylgroup.

However, many non-protein materials do not have sulfhydryl groups andmany proteins do not have a sufficient number of sulfhydryl groups tomake these groups useful for purposes of fluorescence probing.Furthermore, sulfhydryl groups (--SHSH--) oxidized to disulfides(--S--S--) in the presence of air and thereby become unavailable forcovalent attachment to a fluorescence probe.

SUMMARY OF THE INVENTION

In accordance with the present invention, cyanine and relatedpolymethine dyes have been developed having substitutent groups whichare covalently reactive under suitable reaction conditions not only withsulfhydryl groups but also with amine (--NH₂) and hydroxy (--OH) groupsor other groups such as aldehyde (--CHO) groups on proteins and othermaterials for purposes of fluorescence and phosphorescence detection ofthose materials. The present invention offers considerable advantagesover the use of the iodoacetyl cyanine dye of the prior art and itsspecific reactivity with sulfhydryl groups. Amine and hydroxy groups aremore prevalent in proteins and other materials than are sulfhydrylgroups and are more stable. Thereby, when fluorescent cyanine dyes areused for detecting the presence of certain proteins, a strongerfluorescent or phosphorescent light intensity signal will be given offbecause a larger number of dye molecules can be attached to the proteinwhich is being probed. Furthermore, amine and hydroxy groups are moreeasily added to components which it is desired to label, such as polymerparticles, which do not naturally contain either sulfhydryl, amine orhydroxy groups.

This invention also relates to a method wherein luminescent cyanine dyeswhich contain a group which is covalently reactive with amine or hydroxyor other reacting groups are used to label proteins or other materialshaving an amine or hydroxy group or other group capable of reacting withthe dye in a mixture so that the presence and amount of labeled proteinor other material can be detected after the labeled components have beenseparated by chromatographic methods. According to the above citedreferences, apparently the sulfhydryl group was selected for covalentreaction specifically because there are so few of these groups on aprotein molecule and because in some cases the sulfhydryl group plays asignificant role in the function of the protein. Therefore, it waspossible for the authors to attempt to ascertain the specific locationof a sulfhydryl group on a protein structure. Also, in those referencesthe sulfhydryl-specific dye was used as a probe to detect or to producestructural changes in a specific protein. Then, in order to interpret achange in light absorption by the dye or the calcium ion released by dyebinding, it was necessary to know where the probe is bound.

Because there are so few sulfhydryl groups on most protein molecules,those groups may not be sufficiently numerous to provide adequate totalluminescence for detection studies. In contrast, amine and hydroxygroups are significantly more numerous and are widely dispersed on aprotein molecule enabling a fluorescent probe to be attached to multiplesites on the molecule, thereby precluding interpretation of lightabsorption or fluorescence changes, by facilitating the detection of theprotein.

The present invention relates to the labeling with luminescentpolymethine cyanine and related polymethine dyes, such as merocyanineand styryl, of proteins and other materials, including nucleic acids,DNA, drugs, toxins, blood cells, microbial materials, particles, plasticor glass surfaces, polymer membranes, etc., at an amine or hydroxy siteon those materials. The dyes are advantageously soluble in aqueous orother medium in which the labeled material is contained. The presentinvention relates to a two-step labeling process in addition to a singlestep labeling process. In the two-step labeling process, a primarycomponent, such as an antibody, can be labeled at sites thereon,including amine, hydroxy, aldehyde or sulfhydryl sites, and the labeledcomponent is used as the probe for a secondary component, such as anantigen for which the antibody is specific.

In the prior art discussed above, specificity of site of attachment by acyanine probe was achieved by using a probe which is covalently reactivewith a sulfhydryl group. According to the two-step method of the presentinvention, cyanine and related probes can be reacted in a first stepwith amine, aldehyde, sulfhydryl, hydroxy or other groups on a firstcomponent, such as an antibody, and then the antibody can achieve thedesired specificity in a second component, such as an antigen, in asecond or staining step, the specificity being determined by the antigensite of attachment to the antibody.

The present invention is directed also to the luminescent polymethinecyanine and related compounds which contain groups enabling them to becovalently attached to amine, hydroxy, aldehyde or sulfhydryl groups ona target molecule. It is directed to monoclonal antibodies and othercomponents labeled with these luminescent cyanine compounds which arecapable of being probes for antigens. When the target is a type of cell,the present invention can be employed to measure the amount of labeledantibodies which are attached to that type of cell. The measurement canbe made by determining the relative brightness or dimness of theluminescence of the cells.

The present invention can be employed to determine the concentration ofa particular protein or other component in a system. If the number ofreactive groups on a protein which can react with a probe is known, thefluorescence per molecule can be known and the concentration of thesemolecules in the system can be determined by the total luminescenceintensity of the system.

The method can be employed to quantify a variety of proteins or othermaterials in a system by labeling all of a mixture of proteins in thesystem and then separating the labeled proteins by any means, such aschromatographic means. The amount of separated proteins that areluminescent can then be determined. In chromatographic detectionsystems, the location of the dye on the labeled material can beascertained.

This invention can also be employed to determine the number of differentcells which are tagged by an antibody. This determination can be made bytagging a plurality of types of cells in a system, and then separatingthe tagged cells outside of the system. Also, tagged cells can beseparated from nontagged cells outside of the system.

Another embodiment of the present invention comprises a multiparametermethod employing a plurality of luminescent cyanine or related dyesattached respectively to a plurality of different primary components,such as antibodies, each specific for a different secondary component,such as an antigen, in order to identify each of a plurality of saidantigens in a mixture of antigens. According to this embodiment, each ofsaid antibodies is separately labeled with a dye having a differentlight absorption and luminescence wavelength characteristics than thedye used for labeling the other probes. Then, the labeled antibodies areall added to a biological preparation being analyzed containingsecondary components, such as antigens, which can be respectivelystained by particular labeled antibodies. Any unreacted dye materialsmay be removed from the preparation as by washing, if they interferewith the analysis. The biological preparation is then subjected to avariety of excitation wavelengths, each excitation wavelength used beingthe excitation wavelength of a particular conjugated dye. A luminescencemicroscope or other luminescence detection system, such as a flowcytometer or fluorescence spectrophotometer, having filters ormonochrometers to select the rays of the excitation wavelength and toselect the wavelengths of luminescence is employed to determine theintensity of rays of the emission wavelength corresponding to theexcitation wavelength. The intensity of luminescence at wavelengthscorresponding to the emission wavelength of a particular conjugated dyeindicates the quantity of antigen which has been bound to the antibodyto which the dye is attached. In certain cases a single wavelength ofexcitation can be used to excite luminescence from two or more materialsin a mixture where each fluoresces at a different wavelength and thequantity of each labeled species can be measured by detecting itsindividual fluorescence intensity at its respective fluorescencewavelength. If desired, a light absorption detection method can beemployed. The two-step method of the invention can be applied to anysystem in which a primary material conjugated with a dye is used in aluminescence or light absorption detection system to detect the presenceof another material to which the primary material-dye conjugate isdirected. For example, the dye can be conjugated to a fragment of DNA orRNA to form a dye conjugated DNA or RNA fragment which is then directedto a main strand of DNA or RNA to which the piece is complementary. Thesame test method can be employed to detect the presence of anycomplementary main strand of DNA.

The cyanine and related dyes of this invention are especially welladapted for the analysis of a mixture of components wherein dyes of avariety of excitation and emission wavelengths are required becausespecific cyanine and related dyes can be synthesized having a wide rangeof excitation and emission wavelengths. Specific cyanine and relateddyes having specific excitation and emission wavelengths can besynthesized by varying the number of methine groups or by modifying thecyanine ring structures. In this manner, it is possible to synthesizedyes having particular excitation wavelengths to correspond to aparticular excitation light source, such as a laser, e.g., a HeNe laseror a diode laser.

This invention relates to the covalent reaction of highly luminescentand highly light absorbing cyanine and related dye molecules underreaction conditions to amine, hydroxy, aldehyde, sulfhydryl or othergroups on proteins, peptides, carbohydrates, nucleic acids, derivatizednucleic acids, lipids, certain other biological molecules, biologicalcells, as well as to non-biological materials, such as soluble polymers,polymeric particles, polymer surfaces, polymer membranes, glass surfacesand other particles and surfaces. Because luminescence involves highlysensitive optical techniques, the presence of these dye "labels" can bedetected and quantified even when the label is present in very lowamounts. Thus, the dye labeling reagents can be used to measure thequantity of a material that has been labeled. The most useful dyes arehighly light absorbing (ε=70,000 to 250,000 liters per mole centimeter,or higher) and very luminescent and they have quantum yields of at least5% to 80%, or more. The quantities apply to the dyes themselves and tothe dyes conjugated to a labeled material.

An important application for these color labeling reagents is theproduction of luminescent monoclonal antibodies. Monoclonal antibodiesare protein molecules that bind very tightly and very specifically tocertain chemical sites or "markers" on cell surfaces or within cells.These antibodies, therefore, have an enormous research and clinical usefor identifying certain cell types (e.g., HLA classification, T-cellsubsets, bacterial and viral classification, etc.) and diseased cells.In the past, the amount of antibody bound to a cell has been quantifiedby tagging the antibody in various ways. Tagging has been accomplishedwith a radioactive label (radio immunoassay), an enzyme (ELISAtechniques), or a fluorescent dye (usually fluorescein, rhodamine, TexasRed® or phycoerythrin). Most manufacturers and users of clinicalantibody reagents would like to get away from the problems involved inthe use of radioactive tracers so luminescence is considered one of themost promising alternatives. In fact, many companies now marketfluorescein, Texas Red®, rhodamine and phycoerythrin labeled monoclonalantibodies.

In recent years, optical/electronic instrumentation for detectingfluorescent antibodies on cells has become more sophisticated. Forexample, flow cytometry can be used to measure the amount of fluorescentantibody on individual cells at a rate up to 5,000 cells per second.Microscopy and solution fluorescence techniques have also advanced.These instruments can excite fluorescence at many wavelengths of the UV,visible, and near IR regions of the spectrum. Yet most of the usefulfluorescent labeling reagents available today can be excited only in the400-580 nm region of the spectrum. The exceptions are some of thephycobiliprotein-type pigments isolated from marine organisms which canbe covalently attached to proteins and which can be excited at somewhatlonger wavelengths. Therefore, there is a large spectral window rangingfrom 580 to roughly 900 nm where new labeling reagents need to becomeavailable for labeling biological and non-biological materials foranalysis with now available instrumentation. New reagents excitable inthis spectral region would make it possible to perform multicolorluminescence analyses of markers on cells because antibodies withdifferent specificities could each be tagged with a different coloredfluorescent dye. Thus, the presence of several markers could bedetermined simultaneously for each cell analyzed.

This invention also relates to the luminescent (fluorescent orphosphorescent) cyanine, merocyanine and styryl dyes themselves that canbe covalently linked to biological and non-biological materials.Merocyanine and styryl dyes are considered to be related to cyanine dyesfor purposes of this invention. The new labeling reagents themselves,but more particularly when conjugated to a labeled component, can beexcited by light of first defined wavelengths, e.g., by light inwavelength regions of the spectrum ranging from 450 nm to 900 nm.Background fluorescence of cells generally occurs at a lower wavelength.Therefore, the labeling reagents will distinguish over backgroundfluorescence. Particularly of interest are the derivatives that absorblight at 633 nm since they can be excited by inexpensive, intense,stable, long-life, HeNe laser sources. Light of second definedwavelengths that is fluoresced or phosphoresced by the labeled componentcan then be detected. The fluoresced or phosphoresced light generallyhas a greater wavelength than the excitation light. The detection stepcan employ a luminescence microscope having a filter for absorption ofscattered light of the excitation wavelength and for passing thewavelength that corresponds to the luminescence corresponding to theparticular dye label used with the specimen. Such an optical microscopeis described in U.S. patent application Ser. No. 711,065, filed Mar. 12,1985.

Not all cyanine and related dyes are luminescent. However, the dyes ofthis invention include those of the cyanine and related dyes which areluminescent. They are relatively photostable and many are soluble in thereaction solution, preferably a water solution. The conjugated dyesthemselves, but more particularly when such dyes are conjugated to alabeled component, have molar extinction coefficients (ε) of at least50,000, and preferably at least 100,000 liters per mole centimeter. Theextinction coefficient is a measure of the capability of the moleculesto absorb light. The conjugated dyes of this invention have quantumyields of at least 2 percent and preferably at least 10 percent. Inaddition, the conjugated dyes of this invention absorb and emit light inthe 400 to 900 nm spectral range, and preferably in the 600 to 900 nmspectral range.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the monomer absorption spectrum and the dimerabsorption spectrum of a typical cyanine dye dissolved in an aqueousbuffer;

FIG. 2 is a graph showing the absorption spectrum for a particulararylsulfonated dye illustrating that even at high concentrations, thedye has a low tendency to form aggregates in aqueous salt solution;

FIG. 3 is a graph showing the molar dye:protein ratio of an active esterof the new sulfoindodicarbocyanine dye;

FIG. 4 is a graph showing the antibody absorption spectrum of a labelingreagent which does not possess arylsulfonate groups;

FIG. 5 is a graph showing the antibody absorption spectrum of thearylsulfonate cyanine dye labeling reagent of FIG. 3 showing the muchsmaller absorption peak at wavelengths where dimers characteristicallyabsorb; and

FIG. 6 is a graph comparing the quantum yields of sheep immunoglobulinlabeled with the sulfoindodicarbocyanine dye and a protein labeled withan indodicarbocyanine-isothiocyanate reactive dye, showing the change inquantum yield with the change in dye/protein ratios.

ARYLSULFONATED DYES

It has now been found that arylsulfonate or arylsulfonic acidsubstituted dyes as described herein are intrinsically more fluorescentand have improved photostability and water solubility as compared tosimilar dyes without an arylsulfonate or arylsulfonic acid group. Theterm arylsulfonate or arylsulfonic acid as used herein and in the claimsrefers to arylsulfonic acid groups or arylsulfonate groups,interchangeably, wherein said groups are attached to an aromatic ringstructure, including a single ring aromatic structure or a fused ringstructure, such as a naphthalene structure. The single ring aromaticstructure or fused ring aromatic structure can be present in polymethinecyanine, merocyanine or styryl type dyes.

Many dyes with planar molecular structures, including ordinary cyaninedyes, tend to form dimers and higher order aggregates in aqueoussolution, particularly when inorganic salts are also present, as inbuffered solutions and physiological salines. These aggregates usuallyhave absorption bands shifted to the short wavelength side of themonomer absorption and are generally very weakly fluorescent species.The tendency of cyanine dyes to readily form aggregates in aqueoussolution is well known, particularly in the photographic industry (West,W., and Pierce, S., J. Phys. Chem., 69:1894 (1965); Sturmer, D. M.,Spec. Top in Heterocyclic Chemistry, 30 (1974)).

Many dye molecules, and particularly cyanine dye molecules, tend to formaggregates in aqueous solution. It has been found that the arylsulfonatedyes have a minimal tendency to form these aggregates. The arylsulfonatedyes when used to form fluorescent labeling reagents will have a reducedtendency to form aggregates when they are bound at high surfacedensities to protein or other molecules such as antibodies. The tendencyof a particular dye molecule to form aggregates in a salt solution (e.g.150 mM sodium chloride) can be taken as a measure of the tendency of thesame dye molecule to form aggregates on the surface of proteins. It istherefore desirable for dye molecules to have a minimal tendency to formaggregates in aqueous salt solutions. The data shown in FIG. 2 can beused to illustrate that a particular arylsulfonated dye, even at highconcentrations, has a low tendency to form aggregates in aqueous saltsolution.

FIG. 1 shows the monomer absorption spectrum and the dimer absorptionspectrum of a typical cyanine dye dissolved in an aqueous buffer. Thedye used to generate these spectra,N,N'-di-sulfobutyl-indodicarbocyanine, does not possess arylsulfonategroups and readily forms dimers at concentrations even in the submillimolar range. The dimer spectrum was calculated from the spectra ofthe dye at different concentrations (see the method of West, W. andPearce, S., The Dimeric State of Cyanine Dyes, J. Phy. Chem. 69(6),1894-1903 (1965)). At a concentration of 3 millimolar in phosphatebuffered saline solution, the absorbances of the monomer and dimer bandswere about equal.

The spectrum of the improved sulfoindodicarbocyanine,N,N'-diethyl-indodicarbocyanine-5,5'-disulfonic acid, is shown in FIG.2. This dye showed no evidence of aggregation in the saline solution atconcentrations up to 10 millimolar. It is customary to determine theefficiency with which a particular reactive dye couples to a protein,such as an antibody, under defined reaction conditions. The dye testedwas the bis-N-hydroxysuccinimide ester ofN,N'-di-carboxypentyl-indodicarbocyanine-5,5'-disulfonic acid. FIG. 3illustrates that this sulfocyanine dye active ester reacts efficientlywith sheep immunoglobulin in a carbonate buffer at pH 9.2 to formcovalently labeled antibody molecules that have a dye to antibody moleratio ranging from less than 1 to more than 20, depending on therelative dye and antibody concentrations in the reaction solution. Theslope of the linear least squares fit of the data indicates that underthese conditions the labeling efficiency of this dye is about 80%. Insimilar studies fluorescein isothiocyanate (FITC) reacted with about 20%efficiency.

The reactivity of the active ester of the new sulfoindodicarbocyaninedye was investigated by labeling sheep immunoglobulin (IgG). The protein(4 mg/ml) was dissolved in 0.1 molar carbonate buffer (pH 9.2). Aliquotsof the reactive dye dissolved in anhydrous dimethyl formamide were addedto the protein samples to give the original dye protein molar ratios.After thirty minutes the protein was separated from unconjugated dye bygel permeation chromatography (Sephadex® G-50). The resulting molardye:protein ratios were determined spectrophotometrically and are shownin FIG. 3.

At low dye to protein ratios the absorption spectra of the labeledproteins show bands which correspond closely with the spectra of thefree monomeric dye. Antibody molecules which have been heavily labeled(high dye to protein ratios) or which have been labeled with dyes thathave a large tendency to aggregate in aqueous solutions are found oftento have new absorption peaks which appear at shorter wavelengths thanthe absorption bands of the monomeric dye in aqueous solution. Thewavelength of the new absorption peak frequently falls in a region thatis characteristic of the dimer absorption spectrum of the dye (see FIG.1).

More heavily labeled antibodies have higher ratios of short to longwavelength absorption peaks. This shorter wavelength absorption peak canbe seen in FIG. 4 at approximately 590 nm. The longer wavelength peak(at 645 nm) in FIG. 4 is due to monomeric dye molecules bound to theantibody. The labeling reagent used to produce the antibody absorptionspectrum in FIG. 4 (the bis-N-hydroxysuccinimide ester ofN,N'-di-sulfobutyl-indodicarbocyanine-5,5'-acetic acid) does not possessarylsulfonate groups and readily forms dimers in aqueous salt solutionsand on antibodies with which it has reacted. Of key importance,florescence excitation spectra of these antibodies show that excitationof the labeled antibodies at the short wavelength peak does notproportionally produce as much fluorescence as does excitation at thelonger wavelength peak. This observation is in line with the idea thatthe shorter wavelength absorption peak is due to the formation ofnonfluorescent dimers and aggregates on the antibody molecules. We havefound that the aryl sulfonate cyanine dye labeling reagent used inobtaining the data of FIG. 3 does not readily aggregate on the antibodymolecules as judged by the much smaller absorption peak at wavelengthswhere dimers characteristically absorb (see FIG. 5). This is importantbecause antibodies and other proteins labeled with these"nonaggregating" labeling reagents should produce more highlyfluorescent labeled proteins. In fact, the arysulfocyanines do producebrightly fluorescent antibodies even when the average dye per antibodyratio is relatively high (see FIG. 6).

Sheep immunoglobulin (IgG) labeled with a carboxyindodicarbocyanine dyeis shown in FIG. 4. FIG. 5 shows the protein conjugated with the newsulfoindodicarbocyanine dye. The presence of increased dimer (seeFIG. 1) in the former sample is apparent. Although the dye:protein molarratio was approximately the same in both preparations, the proteinrepresented in FIG. 5 was more fluorescent than the sample shown in FIG.4.

In order to have brightly fluorescing antibodies or other proteins thathave been labeled with fluorescent dyes, it is important that theaverage quantum yield per dye molecule on the protein be as high aspossible. It has been generally found that as the surface density of dyemolecules on the protein increases (i.e. the dye to protein ratioincreases), the average quantum yield of the dyes is reduced. Thiseffect has been sometimes attributed to quenching that occurs as aresult of dye-dye interaction on the surface of the more heavily labeledproteins. The formation of non-fluorescent dimers on protein surfacescan certainly contribute to this quenching. FIG. 6 shows that theaverage quantum yield of an arylsulfocyanine dye decreases slowly as thedye/protein ratio increases (curve with diamond symbols). In contrast,the curve having round symbols shows there is a very rapid decrease inthe average quantum yield for the conjugate of a non-arylsulfocyaninedye (N,N'-di-sulfobutyl-indodicarbocyanine-5-isothiocyanate) as thedye/protein ratio increases. Therefore the sulfocyanine dye illustratedin FIG. 6 produces more brightly fluorescing antibodies than the otherdye, especially in the labeling range of 1 to 10 dye molecules perantibody molecule. The average fluorescence quantum yield of individualdye molecules on labeled proteins is a measure of the fluorescencesignal obtainable from these biomolecules. Data from sheepimmunoglobulin (IgG) labeled with the new sulfoindodicarbocyanine dye inphosphate buffered saline solution are shown in the curve having diamondsymbols of FIG. 6. The curve having round symbols of FIG. 6 showsproteins labeled with an indodicarbocyanine-isothiocyanate reactive dye,for comparison. In FIG. 6, the quantum yields at zero dye/protein ratiorepresent the values for the methylamine-adducts of the reactive dyes(free dye) in buffer.

BACKGROUND PROCEDURES

Luminescent probes are valuable reagents for the analysis and separationof molecules and cells and for the detection and quantification of othermaterial. A very small number of luminescent molecules can be detectedunder optimal circumstances. Barak and Webb visualized fewer than 50fluorescent lipid analogs associated with the LDL reception of cellsusing a SIT camera, J. Cell Biol. 90:595-604 (1981). Flow cytometry canbe used to detect fewer than 10,000 fluorescein molecules associatedwith particles or certain cells (Muirhead, Horan and Poste,Bio/Technology 3:337-356 (1985). Some specific examples of applicationof fluorescent probes are (1) identification and separation ofsubpopulations of cells in a mixture of cells by the techniques offluorescence flow cytometry, fluorescence-activated cell sorting andfluorescence microscopy; (2) determination of the concentration of asubstance that binds to a second species (e.g., antigen-antibodyreactions) in the technique of fluorescence immunoassay; (3)localization of substances in gels and other insoluble supports by thetechniques of fluorescence staining. These techniques are described byHerzenberg et al., "Cellular Immunology," 3rd ed., chapt. 22; BlackwellScientific Publications, 1978 (fluorescence-activated cell sorting); andby Goldman, "Fluorescence Antibody Methods" Academic Press, New York,1968 (fluorescence microscopy and fluorescence staining); and inApplications of Fluorescence in the Biomedical Sciences, ed. Taylor etal., Alan Liss Inc., 1986.

When employing fluorescers for the above purposes, there are manyconstraints on the choice of the fluorescer. One constraint is theabsorption and emission characteristics of the fluorescer, since manyligands, receptors, and materials in the sample under test, e.g. blood,urine, cerebrospinal fluid, will fluoresce and interfere with anaccurate determination of the fluorescence of the fluorescent label.This phenomenon is called autofluorescence or background fluorescence.Another consideration is the ability to conjugate the fluorescer toligands and receptors and other biological and non-biological materialsand the effect of such conjugation on the fluorescer. In manysituations, conjugation to another molecule may result in a substantialchange in the fluorescent characteristics of the fluorescer and, in somecases, substantially destroy or reduce the quantum efficiency of thefluorescer. It is also possible that conjugation with the fluorescerwill inactivate the function of the molecule that is labeled. A thirdconsideration is the quantum efficiency of the fluorescer which shouldbe high for sensitive detection. A fourth consideration is the lightabsorbing capability, or extinction coefficient, of the fluorescers,which should also be as large as possible. Also of concern is whetherthe fluorescent molecules will interact with each other when in closeproximity, resulting in self-quenching. An additional concern is whetherthere is nonspecific binding of the fluorescer to other compounds orcontainer walls, either by themselves or in conjunction with thecompound to which the fluorescer is conjugated.

The applicability and value of the methods indicated above are closelytied to the availability of suitable fluorescent compounds. Inparticular, there is a need for fluorescent substances that emit in thelonger wavelength visible region (yellow to near infrared), sinceexcitation of these chromophores produces less autofluorescence and alsomultiple chromophores fluorescing at different wavelengths can beanalyzed simultaneously if the full visible and near infrared regions ofthe spectrum can be utilized. Fluorescein, a widely used fluorescentcompound, is a useful emitter in the green region although in certainimmunoassays and cell analysis systems background autofluorescencegenerated by excitation at fluorescein absorption wavelengths limits thedetection sensitivity. However, the conventional red fluorescent labelrhodamine has proved to be less effective than fluorescein. Texas Red®is a useful labeling reagent that can be excited at 578 nm andfluoresces maximally at 610 nm.

Phycobiliproteins have made an important contribution because of theirhigh extinction coefficient and high quantum yield. Thesechromophore-containing proteins can be covalently linked to manyproteins and are used in fluorescence antibody assays in microscopy andflow cytometry. The phycobiliproteins have the disadvantages that (1)the protein labeling procedure is relatively complex; (2) the proteinlabeling efficiency is not usually high (typically an average of 0.5phycobiliprotein molecules per protein); (3) the phycobiliprotein is anatural product and its preparation and purification is complex; (4) thephycobiliproteins are expensive; (5) there are at present nophycobiliproteins available as labeling reagents that fluoresce furtherto the red region of the spectrum than allophycocyanine, whichfluoresces maximally at 680 nm; (6) the phycobiliproteins are relativelychemically unstable; (7) they photobleach relatively easily; (8) thephycobiliproteins are large proteins with molecular weights ranging from33,000 to 240,000 and are larger than many materials that it isdesirable to label, such as metabolites, drugs, hormones, derivatizednucleotides, and many proteins including antibodies. The latterdisadvantage is of particular importance because antibodies, avidin,DNA-hybridization probes, hormones, and small molecules labeled with thelarge phycobiliproteins may not be able to bind to their targets becauseof steric limitations imposed by the size of the conjugated complex andthe rate of binding of conjugates to targets is slow relative to lowmolecular weight conjugates.

Other techniques involving histology, cytology, immunoassays would alsoenjoy substantial benefits from the use of a fluorescer with a highquantum efficiency, absorption and emission characteristics at longerwavelengths, having simple means for conjugation and being substantiallyfree of nonspecific interference.

OUTLINE OF INVENTION

This invention employs reactive fluorescent arylsulfonated cyanine andrelated dyes having relatively large extinction coefficients and highquantum yields for the purpose of detection and quantification oflabeled components. Fluorescent cyanine and related dyes can be used tolabel biological materials such as antibodies, antigens; avidin,streptavidin, proteins, peptides, derivatized nucleotides,carbohydrates, lipids, biological cells, bacteria, viruses, blood cells,tissue cells, hormones, lymphokines, trace biological molecules, toxinsand drugs. Fluorescent dyes can also be used to label non-biologicalmaterials such as soluble polymers and polymeric and glass particles,drugs, conductor, semiconductor, glass and polymer surfaces, polymermembranes and other solid particles. The component being labeled can bein a mixture including other materials. The mixture, in which the labelreaction occurs, can be a liquid mixture, particularly a water mixture.The detection step can occur with the mixture in a liquid or drycondition, such as a microscope slide.

This invention requires cyanine dyes to be modified by the incorporationinto the cyanine molecule of a reactive group that will covalentlyattach to a target molecule, preferably at an amine or hydroxy site, andin some instances at a sulfhydryl or aldehyde site. This invention alsoemploys modification or use of cyanine and related dye structures toenhance their solubility in the test liquid (1) to make their handlingeasier in labeling reactions, (2) to help prevent aggregation of the dyeon the surface of proteins that are being labeled and (3) to helpprevent nonspecific binding of labeled materials to biological materialsand to surfaces and assay apparatus.

The cyanine and related dyes offer an important advantage over existingfluorescent labeling reagents. First, cyanine and related dyes have beensynthesized that absorb and emit in a region of the spectrum rangingfrom 400 to nearly 1100 nm. Thus reactive derivatives of these dyes canbe made for assays that require simultaneous measurement of a number oflabeled materials. Multicolor (or multiparameter) analysis of this sortmay be desirable for the sake of simplicity, cost effectiveness, or fordetermining ratios of different labeled species on each particle in acomplex mixture of particles (e.g., ratios of antigen markers onindividual blood cells in a complex mixture by multiparameter flowcytometry or fluorescence microscopy). Second, many cyanine and relateddyes strongly absorb and fluoresce light. Third, many cyanine andrelated dyes are relatively photostable and do not rapidly bleach underthe fluorescence microscope. Fourth, cyanine and related dye derivativescan be made which are simple and effective coupling reagents. Fifth,many structures and synthetic procedures are available and the class ofdyes is versatile. Therefore, many structural modifications can be madeto make the reagents more or less water soluble. Their charge can bechanged so they will not perturb the molecule to which they are attachedand so nonspecific binding can be reduced. Sixth, unlike thephycobiliproteins, the cyanine type dyes are relatively small (molecularweight=1,000) so they don't sterically interfere appreciably with theability of the labeled molecule to reach its binding sight rapidly orcarry out its function. Thus cyanine type dye labeling agents offer manypotential advantages. These dyes can be used to selectively label one ormore components in a liquid, especially an aqueous liquid. The labeledcomponents can then be detected by optical or luminescence methods.Alternately, the labeled component can then be used to stain a secondcomponent for which it has a strong affinity, and the presence of thesecond component is then detected by optical or luminescence methods. Inthis case, the dye is reacted with an amine, hydroxy, aldehyde orsulfhydryl group on the labeled component. For example, the labeledcomponent can be an antibody and the stained component for which it hasa strong affinity can be a biological cell, an antigen or a hapten, or abiological cell or particle containing said antigen or hapten. Inanother example, the label component is avidin and the stained componentcan be biotinylated materials. Also, lectins conjugated with polymethinecyanine type dyes can be used to detect and quantify specificcarbohydrate groups. In addition, luminescent cyanine and related dyescan be attached to fragments of DNA or RNA. The labeled fragments of DNAor RNA can be used as fluorescent hybridization probes to identify thepresence and quantity of specific complementary nucleotide sequences insamples containing DNA or RNA. Also, the dye can be attached to ahormone or ligand (such as a hormone, protein, peptide, lymphokine,metabolite) which in turn can be attached to a receptor.

REACTIVE CYANINE DYES ARE DESCRIBED IN PATENTS FOR OTHER USES

Miraha et al. (U.S. Pat. No. 4,337,063), and Masuda et al. (U.S. Pat.No. 4,404,289; U.S. Pat. No. 4,405,711) have synthesized a variety ofcyanine dyes possessing N-hydroxysuccinimide active ester groups. TheMiraha et al. patent, incorporated herein by reference, states, incolumn 2, that the "Spectral sensitizers for photographic use employedfor labelling an antigen or antibody in this invention are well known asspectral sensitizers for photographic light sensitive materials. Cyaninedyes, merocyanine dyes, hemicyanine dyes, styryl dyes, etc. arerepresentative thereof. Detailed disclosure of such spectral sensitizersis provided in . . . Cyanine Dyes and Related Compounds, F. M. Hamer,1964, Interscience Publishers." Masuda et al., U.S. Pat. No. 4,404,289,also incorporated herein by reference, states, in column 10, that"cyanine dyes, merocyanine dyes, hemicyanine dyes, styryl dyes . . . arespecifically described in . . . Cyanine Dyes and Related Compounds, F.M. Hamer (1964), Interscience Publishers". Masuda et al. U.S. Pat. No.4,405,711, incorporated herein by reference, also references, in column10, the F. M. Hamer text for its specific description of cyanine dyes.These patents show that these reagents can be used as photographicsensitizers. The possible fluorescence properties of these reagents arenot mentioned in the patents and, indeed, fluorescence is not requiredfor their process. Most of the dyes mentioned in these patents are onlyweakly fluorescent, they are not especially photostable, and theirsolubility properties are not optimal for many uses that would involvefluorescence detection of labeled materials.

Exekiel et al. (British Patent 1,529,202) have presented numerouscyanine dye derivatives that can be used as covalently reactingmolecules. The Exekiel patent also references, at page 2, the text by F.M. Hamer, supra. The reactive group used in these reagents are azinegroups to which the mono and dichloro-triazine groups belong. TheBritish patent relates to the development and use of these reagents asphotographic film sensitizers. Fluorescence is not required for theprocess and most of the reagents described are not fluorescent. TheBritish patent does not relate to the development and use of reactivecyanine dyes for the purpose of detecting and quantifying labeledmaterials.

DESCRIPTION OF EMBODIMENTS

The present invention pertains to methods for covalently attachingluminescent cyanine and cyanine-type dyes to biological materials,non-biological molecules and macromolecules, and particles in order tomake the material that has been labeled luminescent so that the labeledmaterial can be detected and/or quantified by luminescence detectionmethods.

This invention relates to a method for the detection of a component in aliquid comprising adding to said liquid a dye selected from the groupconsisting of cyanine, merocyanine and styryl dyes which is soluble inthe liquid and contains a substitutent to make it covalently reactivewith amine and hydroxy groups, and possibly to aldehyde and sulfydrylgroups, on said component so that it labels said component. The labeledcomponent is then detected and/or quantified by luminescence or lightabsorption methods. If the labeled component is an antibody, DNAfragment, hormone, lymphokine, or drug, the labeled component can beused to identify the presence of a second component to which it binds,and then the second component can be detected and/or quantified.

Any available luminescence or light absorbing detecting step can beemployed. For example, the detecting step can be an optical detectingstep wherein the liquid is illuminated with light of first definedwavelengths. Light at second defined wavelengths that is fluoresced orphosphoresced by the labeled component is then detected. The detectionalso can be by optical light absorption. For example, the detecting stepcan comprise passing light of first defined wavelengths through theliquid and then ascertaining the wavelength of the light that istransmitted by the liquid.

If desired, the detecting step can comprise chemical analysis tochemically detect attachment of the cyanine or related chromophore tothe component.

The basic structures of cyanine, merocyanine and styryl dyes that can bemodified to create covalent labeling reagents are shown below. ##STR2##

The following are more specific examples of polymethine cyanine typedyes: ##STR3##

In these structures

X and Y are selected from the group consisting of O, S and ##STR4##

Z is selected from the group consisting of O and S; and m is an integerselected from the group consisting of 1, 2, 3 and 4.

In the above formulas, the number of methine groups determines in partthe excitation color. The cyclic azine structures can also determine inpart the excitation color. Often, higher values of m contribute toincreased luminescence and absorbance. At values of m above 4, thecompound becomes unstable. Thereupon, further luminescence can beimparted by modifications at the ring structures. When m=2, theexcitation wavelength is about 650 nm and the compound is veryfluorescent. Maximum emission wavelengths are generally 15-100 nmgreater than maximum excitation wavelengths.

At least one, preferably only one, and possibly two or more of said R₁,R₂, R₃, R₄, R₅, R₆ and R₇ groups in each molecule is a reactive groupfor attaching the dye to the labeled component. For certain reagents, atleast one of said R₁, R₂, R₃, R₄, R₅, R₆ and R₇ groups on each moleculemay also be a group that increases the solubility of the chromophore, oraffects the selectivity of labeling of the labeled component or affectsthe position of labeling of the labeled component by the dye.

In said formulas, at least one of said R₈, R₉ (if any) and R₁₀ (if any)groups comprises at least one sulfonate group. The term sulfonate ismeant to include sulfonic acid because the sulfonate group is merelyionized sulfonic acid.

Reactive groups that may be attached directly or indirectly to thechromophore to form R₁, R₂, R₃, R₄, R₅, R₆ and R₇ groups may includereactive moieties such as groups containing isothiocyanate, isocyanate,monochlorotriazine, dichlorotriazine, mono- or di-halogen substitutedpyridine, mono- or di-halogen substituted diazine, maleimide, aziridine,sulfonyl halide, acid halide, hydroxysuccinimide ester,hydroxysulfosuccinimide ester, imido ester, hydrazine, azidonitrophenyl,azide, 3-(2-pyridyl dithio)-proprionamide, glyoxal and aldehyde.

Specific examples of R₁, R₂, R₃, R₄, R₅, R₆ and R₇ groups that areespecially useful for labeling components with available amino-,hydroxy-, and sulfhydryl groups include: ##STR5## where at least one ofQ or W is a leaving group such as I, Br, Cl, ##STR6##

Specific examples of R₁, R₂, R₃, R₄, R₅, R₆ and R₇ groups that areespecially useful for labeling components with available sulfhydrylswhich can be used for labeling antibodies in a two-step process include:##STR7## where Q is a leaving group such as I or Br, ##STR8## where n is0 or an integer.

Specific examples of R₁, R₂, R₃, R₄, R₅, R₆ and R₇ groups that areespecially useful for labeling components by light-activitated crosslinking include: ##STR9##

For the purpose of increasing water solubility or reducing unwantednonspecific binding of the labeled component to inappropriate componentsin the sample or to reduce the interactions between two or more reactivechromophores on the labeled component which might lead to quenching offluorescence, the R₁, R₂, R₃, R₄, R₅, R₆ and R₇ groups can be selectedfrom the well known polar and electrically charged chemical groups.Examples are --E--F where F is hydroxy, sulfonate, sulfate, carboxylate,substituted amino or quaternary amino and where E is a spacer group suchas --(CH₂)_(n) --where n is 0, 1, 2, 3 or 4. Useful examples includelower alkyls and alkyl sulfonate; --(CH₂)₃ SO₃ ⊕ and --(CH₂)₄ --SO₃ ⊕.

The polymethine chain of the luminescent dyes of this invention may alsocontain one or more cyclic chemical groups that form bridges between twoor more of the carbon atoms of the polymethine chain. These bridgesmight serve to increase the chemical or photostability of the dye andmight be used to alter the absorption and emission wavelength of the dyeor change its extinction coefficient or quantum yield. Improvedsolubility properties may be obtained by this modification.

In accordance with this invention the labeled component can beantibodies, proteins, peptides, enzyme substrates, hormones,lymphokines, metabolites, receptors, antigens, haptens, lectins, avidin,streptavidin, toxins, carbohydrates, oligosaccharides, polysaccharides,nucleic acids, deoxy nucleic acids, derivatized nucleic acids,derivatized deoxy nucleic acids, DNA fragments, RNA fragments,derivatized DNA fragments, derivatized RNA fragments, natural drugs,virus particles, bacterial particles, virus components, yeastcomponents, blood cells, blood cell components, biological cells,noncellular blood components, bacteria, bacterial components, naturaland synthetic lipid vesicles, synthetic drugs, poisons, environmentalpollutants, polymers, polymer particles, glass particles, glasssurfaces, plastic particles, plastic surfaces, polymer membranes,conductors and semiconductors.

A cyanine or related chromophore can be prepared which when reacted witha component can absorb light at 633 nm and the detecting step can employa helium neon laser that emits light at this wavelength of the spectrum.Also, a cyanine or related dye can be prepared which when reacted with acomponent can absorb light maximally between 700 nm and 900 nm and thedetecting step can employ a laser diode that emits light in this regionof the spectrum.

SELECTIVITY

The reactive groups listed above are relatively specific for labelingparticular functional groups on proteins and other biological ornon-biological molecules, macromolecules, surfaces or particles providedthat appropriate reaction conditions are used, including appropriate pHconditions.

PROPERTIES OF THE REACTIVE CYANINE, MEROCYANINE AND STYRYL DYES ANDTHEIR PRODUCTS

The spectral properties of the dyes of this invention are notappreciably altered by the functionalization described in thisspecification. The spectral properties of labeled proteins and othercompounds are also not very different from the basic dye molecule thathas not been conjugated to a protein or other material. The dyesdescribed in this invention alone or conjugated to a labeled materialgenerally have large extinction coefficients (ε=100,000 to 250,000),have quantum yields as high as 0.4 in certain cases, and absorb and emitlight in the spectral range of 400 to 900 nm. Thus, they are especiallyvaluable as labeling reagents for luminescence detection.

OPTICAL DETECTION METHODS

Any method can be employed for detecting a labeled or stained component.The detecting method can employ a light source that illuminates themixture containing the labeled material with light of first definedwavelengths. Known devices are employed that detect light at secondwavelengths that is transmitted by the mixture or is fluoresced orluminesced by the mixture. Such detection devices include fluorescencespectrometers, absorption spectrophotometers, fluorescence microscopes,transmission light microscopes and flow cytometers, fiber optic sensors,and immunoassay instruments.

The method of this invention can also employ chemical analysis methodsto detect attachment of the dye to the labeled component or components.Chemical analysis methods can include infrared spectrometry, NMRspectrometry, absorption spectrometry, fluorescence spectrometry, massspectrometry and chromatographic methods.

Arylsulfonation of intermediates useful to form styryl dyes wasperformed according to Examples A and B which were set forth in BelgiumPatent No. 669,003 (1965) relating to solubilized styryl dyes, whichpatent is cited by the Sturmer reference, supra. at 553. This standardsulfonation procedure may be used for the arylsulfonation of the presentinvention.

EXAMPLE A

2-methyl-8-sulfonaphth[1,2-d]oxazole 48 g (1 mole) of1-amino-2-naphthol-4-sulfonic acid and 80 ml of pyridine are mixed andthis mixture is then dissolved by adding 30 ml water. The amber coloredsolution is then heated gently in a heating jacket and 360 ml of aceticanhydride are added in small fractions. During this addition, a largeamount of heat is evolved, causing the mixture to reflux vigorously.After the addition of the acetic anhydride is complete, the mixture isheated under reflux for 2 hours. By removing the solvents under reducedpressure, a thick syrup is obtained which is dissolved in 500 ml water.The aqueous solution is then rendered strongly acid to universalindicator paper by adding concentrated sulfuric acid, with stirring. Theproduct thereupon precipitated from the solution in the form of a whitesolid. The solid is collected by filtration, washed with water anddried. After two recrystallizations from ethyl alcohol, 35 g. (67%) of awhite solid, melting above 320° C., are obtained.

EXAMPLE B Monosulfonated 1,1,2-trimethyl-1H-benz[e]indole ##STR10##

5 g (1 mole) of 1,1,2-trimethyl-H-benz[e]indole and 50 ml ofconcentrated sulfuric acid are mixed, after which the mixture is heatedat 180° C. for 30 minutes. The mixture is then cooled, poured onto 100 gof ice and neutralized with 25 ml of 25% strength sodium hydroxide,after which the crystalline product is collected by filtration, washedwith acetone and dried. After two recrystallizations from water, 5.4 g(80%) of pure product, melting above 310° C., are obtained.

EXAMPLE 1 Effect Of pH On The Conjugation Of SulfoindodicarbocyanineWith Protein

Samples of sheep gamma-globulin (4 mg/ml) in 0.1M carbonate buffers (pH8.5, 8.9 and 9.4) were mixed at room temperature with a 10 fold molarexcess of the following sulfoindodicarbo-cyanine active-ester (m=2).##STR11## At appropriate times, ranging from 5 seconds to 30 minutes,protein samples were separated from non-covalently attached dye by gelpermeation chromatography on Sephadex® G-50. Maximum labeling of theprotein occurred after 10 minutes, yielding final dye/protein moleratios of 5.8, 6.4 and 8.2 for the samples incubated at pH 8.5, 8.9 and9.4, respectively. The times required to produce a dye/protein ratio of5 and the quantum yields of the products at the different pH levels areshown in the table below:

    ______________________________________                                        pH            time (sec.)                                                                             OY                                                    ______________________________________                                        8.5           115       0.09                                                  8.9           53        0.09                                                  9.4           6.5       0.17                                                  ______________________________________                                    

These data indicate that protein labeling with this dye is better at pH9.4 than below 9. In the higher pH buffer the conjugation reaction wasvery rapid, but the labeling efficiency was excellent and the productwas more fluorescent. The quantum yield value represents the averagequantum yield per dye molecule on the labeled protein.

EXAMPLE 2 Sulfoindocarbocyanine Active Ester Conjugation With Protein

Sheep gamma-globulin (1 mg/ml) dissolved in pH 7.4 phosphate bufferedsaline (PBS) was adjusted to pH 9.4 using 0.1M sodium carbonate. Cyaninedye labeling agent (structure in Example 1, m=1) was added to aliquotsof this protein solution to give various mole ratios of dye/protein.After 30 minutes incubation at room temperature the mixtures wereseparated by Sephadex® G-50 gel permeation chromatography eluting withPBS. The mole ratio of dyes covalently attached to the proteins in theproducts were 1.2, 3.5, 5.4, 6.7 and 11.2 for initial dye/protein ratiosof 3, 6, 12, 24 and 30, respectively.

EXAMPLE 3 Labeling AECM-Dextran With A Sulfoindodicarbocyanine

N-Aminoethyl-carboxamidomethyl (AECM) dextran containing an average of16 amino groups per dextran molecule was synthesized from dextran,average MW 70000 (Inman, J. K., J. Immunol. 114: 704-709 [1975]). Aportion of the AECM-dextran (1 mg/250 μl) dissolved in 0.1M carbonatebuffer, pH 9.4 was added to 0.2 mg of the sulfoindodicarbocyanine activeester (structure in Example 1, m=2) giving a dye/protein mole ratio of10. The mixture was stirred for 30 minutes at room temperature. Thedextran was then separated from nonconjugated dye by Sephadex® G50 gelpermeation chromatography using ammonium acetate (50 mM) as elutionbuffer. An average of 2.2 dye molecules were covalently linked to eachdextran molecule.

EXAMPLE 4 Sulfoindodicarbocyanine Active Ester Labeling of SpecificAntibody

Sheep gamma-globulin specific against murine lgG (1 mg/ml) in 0.1Mcarbonate buffer (pH 9.4) was mixed with sulfoindodicarbocyanine activeester (structure in Example 1, m=2) at a ratio of 8 dyemolecules/protein molecule. After incubating 30 minutes at roomtemperature, the labeling mixture was separated by gel filtration overSephadex® G-50 equilibrated with phosphate buffered saline (pH 7.4). Therecovered protein contained an average of 4.5 dye molecules covalentlyattached to each protein molecule.

EXAMPLE 5 Staining and Microscopic Visualization Of Human LymphocytesWith Sulfoindodicarbocyanine Dye Conjugated To Sheep Anti-mouse-lgGAntibody

Freshly isolated peripheral blood lymphocytes were treated at 0° C. for30 minutes with mouse anti-beta2-microglobulin (0.25 μg 10⁶ cells). Thecells were washed twice with DMEM buffer and were then treated with thesulfoindodicarbocyanine-labeled sheep anti-mouse-lgG antibody (1 μg/10⁶cells). After a 30 minute incubation at 0° C., the excess antibody wasremoved by centrifuging the cells and the cells were again washed twicewith DMEM buffer. Aliquots of the cells were fixed on slides foranalysis by fluorescence microscopy. Under the microscope thelymphocytes on the slide were illuminated with light at 610-630 nm andthe fluorescence at 650-700 nm was detected with a COHU red sensitiveintensified television camera attached to an image digitizer andtelevision monitor. The cells stained by this method showed fluorescenceunder the microscope. In a control experiment, use of the primary mouseanti-beta2-microglobulin antibody was omitted but the staining andanalysis was otherwise carried out as described above. The controlsample showed no fluorescence under the microscope indicating that thesulfoindocyanine-labeled sheep anti-mouse antibody does not givesignificant nonspecific binding to lymphocytes.

We claim:
 1. A water soluble luminescent dye consisting of:a cyaninehaving the structure ##STR12## wherein: the dotted lines each representcarbon atoms necessary for the formation of one ring to three fusedrings having 5 to 6 atoms in each ring and said R₃, R₄, R₈ and R₉ groupsare attached to said rings;X and Y are each CH₃ --C--CH₃ ; m is aninteger selected from the grouping consisting of 1, 2 and 3; at leastone of said R₁, R₂, R₃, R₄ and R₇ groups reacts with amino, hydroxy orsulfhydryl nucleophiles and is a reactive moiety selected from the groupconsisting of ##STR13## wherein Q is Br or Cl, and n=0, 1, 2, 3, 4, 5,6, 7 or 8 for R₃, R₄ and R₇, and n=1, 2, 3, 4, 5, 6, 7 or 8 for R₁ andR₂ ; and when any one of said R₃, R₄, and R₇ groups is not selected fromone said reactive moiety, said remaining R₃, R₄, and R₇ are hydrogen orE--F, a polar group wherein E is a spacer group having the structure--(CH₂)_(n) -- and n of said spacer group=0, 1, 2, 3, or 4, and F ishydroxy, sulfonate, sulfate, carboxylate, or a lower alkyl substitutedamino; and when any one of said R₁ and R₂ groups is not selected fromone said reactive moiety, said remaining R₁ or R₂ are a lower alkyl orsaid E--F and n of said spacer group=1, 2, 3, or 4; at least one said R₈and R₉ groups is selected from the group consisting of a sulfonic acidand a sulfonate moiety attached directly to said ring for conferringimproved luminescence to said dye when in use as compared to a dye whichdoes not have said at least one sulfonic acid or sulfonate moiety; andwhen one of said R₈ or R₉ group is not a sulfonic acid or a sulfonate,said remaining R₈ or R₉ group is a hydrogen.
 2. The luminescent dye ofclaim 1 wherein said cyanine has the structure: ##STR14##
 3. Theluminescent dye recited in claim 1 wherein the cyanine has thestructure: ##STR15## wherein R₁₀ is selected from the group consistingof said reactive moiety wherein n=0, 1, 2, 3, 4, 5, 6, 7 or 8, hydrogenand sulfonic acid or sulfonate moiety.
 4. A water soluble luminescentdye consisting of a cyanine having the structure ##STR16## wherein: Xand Y are CH₃ --C--CH₃ ;m is an integer selected from the groupconsisting of 1, 2 and 3; at least one of said R₁, R₂, R₃, R₄, R₇, R₁₀and R₁₁ groups reacts with amino, hydroxy or sulfhydryl nucleophiles andis a reactive moiety selected from the group consisting of: ##STR17##wherein Q is Br or Cl, and n=0, 1, 2, 3, 4, 5, 6, 7 or 8 for R₃, R₄, R₇,R₁₀ and R₁₁, and n=1, 2, 3, 4, 5, 6, 7 or 8 for R₁ and R₂ ; and when anyone of said R₃, R₄, and R₇ groups is not selected from one said reactivemoiety, said remaining R₃, R₄, and R₇ are hydrogen or E--F, a polargroup wherein E is a spacer group having the structure --(CH₂)_(n) --and n=0, 1, 2, 3, or 4, and F is hydroxy, sulfonate, sulfate,carboxylate, or lower alkyl substituted amino; and when any one of saidR₁ and R₂ groups is not selected from one said reactive group, saidremaining R₁ and R₂ are lower alkyl or said E--F and n=1, 2, 3, or 4;said R₈ and R₉, and said R₁₀ and R₁₁ groups when said R₁₀ and R₁₁ groupsare not said reactive moiety, are selected from the group consisting ofhydrogen and a sulfonic acid or sulfonate moiety, wherein at least oneof said R₈, R₉, R₁₀ and R₁₁ groups is one of said sulfonic acid or saidsulfonate moiety attached directly to a ring of said dye for conferringimproved luminescence to said dye when in use as compared to a dye whichdoes not have said at least one sulfonic acid or sulfonate moiety.
 5. Awater soluble luminescent dye consisting of: ##STR18## wherein X and Yare CH₃ --C--CH₃ ; m is an integer selected from the group consisting of1, 2 and 3;at least one of said R₁ and R₂ is ##STR19## and the other isselected from the group consisting of ##STR20## --CH₂ CH₂ OH,--(CH₂)_(k) SO₃ where n=1, 2, 3, 4, 5, 6, 7 or 8 and k=2, 3, 4 or 5; andat least one of said R₈ and R₉ groups is a sulfonate moiety attacheddirectly to a ring of said dye for conferring improved luminescence tosaid dye when in use as compared to a dye which does not have at leastone sulfonic acid or sulfonate moiety; and when one of said R₈ and R₉group is not said sulfonate moiety, said remaining R₈ or R₉ group is ahydrogen.
 6. A water soluble luminescent dye consisting of: ##STR21##wherein X and Y are CH₃ --C--CH₃ ; m is an integer selected from thegroup consisting 1, 2 and 3;at least one of said R₁ and R₂ is ##STR22##and the other is selected from the group consisting of ##STR23## --CH₂CH₂ OH, --(CH₂)_(k) SO₃ where n=1, 2, 3, 4, 5, 6, 7, or and k=2, 3, 4,or 5; at least two of said R₈, R₉,R₁₀ and R₁₁ groups are a sulfonatemoiety attached directly to a ring of said dye for conferring improvedluminescence to said dye when in use as compared to a dye which does nothave at least one sulfonic acid or sulfonate moiety; and when any one ofsaid R₈, R₉, R₁₀ and R₁₁ groups is not said sulfonate moiety, it is ahydrogen.
 7. A water soluble luminescent dye consisting of: ##STR24##wherein X and Y are CH₃ --C--CH₃ ; m is an integer selected from thegroup consisting of 1, 2, and 3;at least one of said R₁ and R₂ is##STR25## and the other is selected from the group consisting of##STR26## where n=1, 2, 3, 4, 5, 6, 7 or 8 and k=2, 3, 4 or 5; at leasttwo of said R₈, R₉ and R₁₀ groups are a sulfonate moiety attacheddirectly to a ring of said dye for conferring improved luminescence tosaid dye when in use as compared to a dye which does not have at leastone sulfonic acid or sulfonate moiety; and when any one of said R₈, R₉or R₁₀ is not said sulfonate moiety, it is a hydrogen.
 8. A watersoluble luminescent dye consisting of: ##STR27## wherein m is an integerselected from the group consisting of 1 and 2 and n=5.